In spinal cord injuries, axons are cut from the neuronal cell bodies, and lack of axon regeneration is the principal cause of no or limited functional recovery in the central nervous system (CNS, including the brain and the spinal cord). While earlier studies emphasized the importance of neuron-extrinsic mechanisms, recent development in the field highlighted the central role of neuron-intrinsic control of axon growth and regeneration after CNS injury. Meanwhile, it is increasingly clear that targeting one molecule or pathway at a time is unlikely to bring about functionally meaningful axon growth and regeneration. miRNAs are small non-coding RNAs that post-transcriptionally regulate protein synthesis. One miRNA can regulate the expression of multiple proteins in multiple pathways, sometimes related in function. Here we propose to explore the role of miRNAs in spinal axon sprouting and regeneration after CNS injury. We hypothesize that in neurons some miRNAs are growth inhibitory while others are growth promoting. Reversal of the expression of growth inhibitory and promoting miRNAs after CNS injury may allow neurons to enter a more regenerative state, thus promoting axon growth and regeneration. In Aim 1, we will systematically profile miRNA expression in corticospinal neurons and axons before and after spinal cord injury and compare these data to miRNA expression in postnatal neurons that still possess significant axon growth ability. A comparison of miRNA expression profiles in neurons of different levels of regenerative abilities may provide important clues to the pattern and identiy of the miRNAs that may positively or negative regulate axon growth. The expression profiling will be considered together with target predictions to narrow down the candidate miRNAs to be functionally tested. In Aim 2, we will assess the effect of manipulating candidate miRNAs by overexpression or inhibition on axon growth in vitro using microfluidic chambers. We will use adeno-associated virus (AAV) to deliver miRNAs or inhibitory sponge constructs. These experiments will help us to further narrow down the number of candidates for in vivo studies. In Aim 3, we will assess the effect of manipulating candidate miRNAs on corticospinal axon sprouting and regeneration using pyramidotomy and dorsal hemisection spinal cord injury respectively. Together, these studies will start to assess the role and therapeutic potential of miRNAs for promoting axonal repair after CNS injury. The unique feature of miRNAs regulating multiple molecular targets and pathways simultaneously renders them attractive therapeutic targets and tools for diseases and injuries. This proposal represents an early step in the application of miRNA biology to axonal repair after CNS injury.